I. Molecular Biology of Trichoderma
Filamentous fungi are lower eukaryotes widely used in biotechnology to make various fermentation products. Fungi secrete many industrially important enzymes such as glucoamylase, proteases, lactase, pectinases and glucose oxidase.
Filamentous fungi have a number of advantages for biotechnology. They generally produce high amounts of proteins, cultivation in large scale is not complicated and separation of the mycelium from the culture liquid after the fermentation is easy. For example, certain hypercellulolytic strains of the filamentous fungi Trichoderma are capable of secreting over 40 grams of cellulases per liter of culture medium (Durand, H. et al., in Biochemistry and Genetics of Cellulase Degradation, Academic Press, San Diego, 135-151 (1988); Durand, H. et al., Enzyme Microbiol. Technology 10:341-346 (1988)). In addition, there are indications that Trichoderma does not hyperglycosylate proteins as Saccharomyces yeast does (EP 215,594; Penttila, M., Construction and Characterization of Cellulolytic Yeasts, VTT Publication Series No. 39, VTT Technical Research Center, Espoo, Finland, (1987)).
The mesophilic imperfect fungus Trichoderma reesei (now also known as Trichoderma pseudokoningii Rafai and formerly T. viride) (Can. J. Bot. 62:924; Simmons, E. G., In: Abstracts of Second International Mycological Congress, Tampa, Fla., Aigelow, H. E., et al., (eds.), p. 618 (1977)) produces substantial amounts of enzymes needed in the conversion of cellulosic biomass and is probably the most widely investigated of all cellulase-producing organisms.
For hydrolysis of cellulose to glucose, three types of enzyme activity are needed: randomly cleaving endoglucanases (1,4,-.beta.-D-glucan glucanohydrolase, EC 3.2.1.4) which usually attack substituted soluble substrates and show no activity to crystalline cellulose; cellobiohydrolase (1,4-.beta.-D-glucan cellobiohydrolase, EC 3.2.1.91) capable of degrading crystalline cellulose but having no activity towards derivatized cellulose and .beta.-glucosidase (.beta.-D-glucoside glycohydrolase, EC 3.2.1.21) attacking cellobiose and cello-oligosaccharides to yield glucose. Synergistic action between some of these enzymes has been demonstrated (Berghem, L. E. R., et al., Eur. J. Biochem. 97:21-30 (1973); Gong, C. S., et al., Adv. Chem. Ser. 181:261-287 (1979); Fagerstam, L. G., et al., FEBS Letters 119:97-100 (1980)).
Fungal cellulases have been purified and characterized and all three main types of enzymes have been shown to occur in multiple forms (Enari, T. M., In: Microbial Enzymes and Biotechnology, Fogarty, W. M. (ed.), Applied Science Publishers, London and New York, pp. 183-223 (1983)). Two immunologically distinctive cellobiohydrolases, CBH I and CBH II have been detected from the culture medium of T. reesei (Fagerstam, L. G., et al., FEBS Letters 119:97-100 (1980); Gilbert, I. G., et al., Ann. Reports on Fermentation Processes 6:323-358 (1983)). Five to eight electrophoretically distinct endoglucanases have been reported, many of them showing varying substrate specificities (Shoemaker, S. P., et al., Biochim. Biophys. Acta 523:133-146 (1978); Shoemaker, S. P., et al., Biochim. Biophys. Acta 523:147-161 (1978); Bissett, F. H., J. Chromatog. 178:517-523 (1979); Farkas, V. A., et al., Biochem. Biophys. Acta 706:105-110 (1982)). Characterization of two extracellular .beta.-glucosidases has been reported (Enari, T. M., et al., In: Proceedings of 2nd International Symposium on Bioconversion and Biochemical Engineering, Ghose, T. K. (ed.), Indian Institute of Technology, New Delhi, 1:87-95 (1980)).
Intensive strain development using the direct approach of mutation and screening has successfully produced several high-yielding T. reesei mutant strains (Bailey, M. J., et al., Enzyme Microb. Technol. 3:153-157 (1981); Andreotti, R., et al., In: Proceedings of 2nd International Symposium on Bioconversion and Biochemical Engineering, Ghose, T. K. (ed.), Indian Institute of Technology, New Delhi, 1:353-388 (1980); Farkas, V., et al., Folia Microbiol. 26:129-132 (1981); Montenecourt, B. S., et al., Adv. Chem. Ser. 181:289-301 (1979); Mandels, M., et al., Appl. Microbiol. 21:152-154 (1971); Gallo, B. J., et al., Biotechnol. Bioengineer. Symp. 8:89-101 (1979); Warzywoda, M. et al., Biotechnol. Lett 5:243-246 (1983); Montenecourt, B. S., et al., Appl. Environ. Microbiol. 34:777-782 (1977); Sheir-Neiss, G., et al., Appl. Microbiol. Biotechnol. 20:46-53 (1984); Shoemaker, S. P., et al., In: Trends in the Biology of Fermentations for Fuels and Chemicals, Hollaender, A. E. (ed.), Plenum Press, pp. 89-109 (1981); Nevalainen, K. M. H., et al., Enzyme Microb. Technol. 2:59-60 (1980)). The amount of extracellular protein produced by the best T. reesei routants is more than 50% of the total cell protein. A major part of the secreted protein comprises cellulases among which the CBH I component is most abundant, representing up to 60% of the secreted cellulase proteins.
The gene for CBH I has been cloned by Shoemaker et al. (Shoemaker, S., et al., Bio/Technology 1:691-695 (1983)) and Teeri et al. (Teeri, T., et al., Bio/Technology 1:696-699 (1983)) and the entire nucleotide sequence of the gene has been published (Shoemaker, S., et al., Bio/Technology 1:691-696 (1983)). From T. reesei, the gene for the major endoglucanase EG I has also been cloned and characterized (Penttila, M., et al., Gene 45:253-263 (1986); Patent Application EP 137,280; Van Arsdell, J. N. V., et al., Bio/Technology 5:60-64 (1987)). Other isolated cellulase genes are cbh2 (Teeri et al., Gene 51:43-52 (1987); Patent Application WO 85/04672; Chen, C. M., et al., Bio/Technology 5:274-278 (1987)) and egl2, previously called egl3 (Saloheimo, M., et al., Gene 63:11-21 (1988).
The molecular biology of industrially important filamentous fungi is in general not well known. This is partly due to the lack of the sexual reproduction cycle and/or genetic transformation system. Transformation systems have been developed for a number of filamentous fungi, for example, Neurospora crassa (Case, M., et al., Proc. Natl. Acad. Sci. USA 76:5259-5263 (1979)), A. nidulans (Ballance, J., et al., Biochem. Biophys. Res. Commun. 112:284-289 (1983); Tilburn, J., et al., Gene 26:205-221 (1983); Yelton, M., et al., Proc. Natl. Acad. Sci. USA 81:1470-1474 (1984)), and A. niger (Kelly, J. M., et al., EMBO Journal 4:475-479 (1985)), A. oryzae (Gomi, K. et al., Agric. Biol. Chem. 51:2549:2555 (1987) and Mattern, I. E. et al., Mol. Gen. Genet. 210:460-461 (1987) and A. awamori (Berka, R. M. et al., Gene 86:153-162 (1990) generally having their basis in complementation of the mutant host by respective functional gene carried by the vector molecule. However, of these fungi only A. niger, A. awamori and A. oryzae are of industrial interest at the moment.
In the classification of fungi, the genus Aspergillus is included in the class Ascomycetes, sub-class Euascomycetes. Euascomycetes are divided into three groups, Plectomycetes, Pyrenomycetes and Discomycetes on the basis of the fruiting bodies. The most important genera are Aspergillus and Penicillium (Doi, Y., Bull. Nat. Sci. Mus. Tokyo 11:185-189 (1968)). Trichoderma, instead, is classified as a member of Fungi imperfecti. Fungi imperfecti is a catch-all category of fungi which have no sexual reproduction or obvious affinities with sexually reproducing genera, such as the highly characteristic Aspergillus. Although Trichoderma has been reported to possess a poorly defined sexual stage being an imperfect state of the perfect ascomycete species Hypocrea (Beja da Costa, M., et al., Biotechnol. Biogen. 22:2429-2432 (1980)), the genera Aspergillus and Trichoderma are clearly to be considered taxonomically very different. It has also been shown that the argB gene from Aspergillus nidulans and the pyr4 gene from Neurospora crassa do not hybridize, under non-stringent conditions, to the respective Trichoderma genes, thus supporting the idea that Trichoderma is evolutionarily quite distinct from other Ascomycetes.
Due to its exceptional ability to secrete proteins into the growth medium Trichoderma reesei is a good candidate as a possible host for the production of proteins. However, genetic studies of T. reesei have so far been directed almost exclusively to improving the cellulose-producing properties of the fungus and the only technique used for the development of hypercellulotic Trichoderma strains has been traditional mutagenesis and screening.
In the fungal transformation systems first developed, namely for Aspergillus and Neurospora, transformation is carried out using protoplasts. The transforming DNA is usually integrated into the host genome. To provide a selection system for identifying stable transformants the vector system must carry a functional gene (a selection marker) which either complements a corresponding mutation of the host genome or supplies an activity, usually an enzyme, required for the growth of the prototrophic strain on a particular growth medium.
Durand et al., in Biochemistry and Chemistry of Cellulose Degradation, Aubert et al., eds., Academic Press, 1988, pp. 135-151 reported a transformation system for Trichoderma which was based on phleomycin resistance. However the transformants were unstable unless restreaked or kept on phleomycin plates for a long time (Berges, T. et al., in Abstracts, Tricel 89, International Symposium on Trichoderma Cellulases, Vienna, 1989, p.21, also reports a transformation system for Trichoderma). However, the Berges system also results in unstable transformants.
II. Immunoglobulins
Immunoglobulins consist of two identical light chains and two identical heavy chains linked by sulphur bridges. The polypeptide chains of immunoglobulins are formed from successive domains, which can fold up independently. Light chains consist of two domains, heavy chains usually four-five domains. The ability of immunoglobulins to recognize and specifically bind their antigens is due to differences in the variable domains which reside at the N-terminal ends of both the light and heavy chains.
An antibody molecule can be proteolysed into two parts, the so-called Fc and Fab fragments. The Fab fragment includes the sites from the variable domains of both the light and heavy chains which are responsible for the identification and binding of antigens.
The domains of the Fc fragment play roles in the activation of the immune response in a cell. Biochemical tests have shown that the Fc fragment is not necessary for antigen binding.
Through genetic engineering it is possible to produce just the Fab antigen-binding sites of antibodies (Better, M., et al., Science, 240:1041-1042 (1988); Cabilly, S., Gene 85:552-553 (1989). By combining independently folding domains of antibodies in various ways it has also been possible to produce chimeric antibodies (Morrison, S. L., EP 173,494; Cabilly, S., EP 125,023). It has, for example, been possible to combine human antibody domains with variable mouse antibody domains thus changing the antibody spectrum. Genetic engineering also allows the production of single-chain antibodies, in which an antigen-binding site is formed by only one polypeptide chain.
Monoclonal antibodies are produced in hybridoma and myeloma tissue cultures, often with human or mouse hybridoma cell lines. The methods are fairly expensive and the yields of monoclonal antibodies from human hybridoma cell lines are relatively low (1 .mu.g/ml for human hybridomas compared to 100 .mu.g/ml for mouse hybridomas).
In addition, most human monoclonal antibodies obtained in cell culture are of the IgM type. When it is desirable to obtain human monoclonals of the IgG type, it has been necessary to utilize cell sorting techniques to separate the few cells which have switched to producing antibodies of the IgG or other type from the majority producing antibodies of the IgM type. This greatly increases the expense and the yield of human IgG type monoclonal antibodies is even lower than that of the IgM type.
Efforts have been made to develop better methods, by transferring antibody genes into a host organism, such as the bacterium Escherichia coli (Better, M., et al., Science, 240:1041-1042 (1988)) and the yeast Saccharomyces cerevisiae (Horwitz, A. H., et al., Proc. Natl. Acad. Sci. USA 85:8678-8682 (1988). These have been used for the production of Fab sites. E. coli has also been used for the production of single chain antibodies. However, yield of the active product is still low, probably, for the most part, because of the naturally poor protein secretion capacity of the organisms. Thus, there remains a need for an economical manner in which to produce the amounts of immunoglobulins, and especially monoclonal antibodies needed for industrial and medical applications.